Bacillopeptidase C, a new alkaline protease and its production by cultivating bacillus bacteria

ABSTRACT

A novel protease named Bacillopeptidase C and having strong anti-inflammatory activity is produced by Bacillus sp. No. 794 when this microorganism is cultivated. The Bacillopeptidase C differs from other alkaline proteases in isoelectric point and behaviors to inhibitors.

United States Patent [1 1 Hidaka et al.

[4 1 Sept. 16, 1975 BACILLOPEPTIDASE C, A NEW ALKALINE PROTEASE AND ITS PRODUCTION BY CULTIVATING BACILLUS BACTERIA Inventors: Hidemasa Hidaka, Yokohama;

Kenji Yoshida, Kawasaki; Uichi Shibata, Tokyo; Yasukatsu Yuda, Yokohama; Tomizo Niwa, Kawasaki; Hitoshi Goi, Kawasaki; Shinji Miyado, Yokohama; Yujiro Yamada, Yokohama; Takemi Koeda, Yokohama; Kazuo Saito, Fujisawa, all of Japan Assignee: Meiji Seika Co., Ltd., Japan Filed: Aug. 29, 1973 Appl. No.: 392,426

Foreign Application Priority Data Sept. 2, 1973 Japan 47-88137 Primary Examiner-Lionel M. Shapiro Attorney, Agent, or Firm-McGlew and Tuttle [5 7] ABSTRACT A novel protease named Bacillopeptidase C and hav ing strong anti-inflammatory activity is produced by Bacillus sp. No. 794 when this microorganism is cultivated. The Bacillopeptidase C differs from other alkaline proteases in isoelectric point and behaviors to inhibitors.

10 Claims, 5 Drawing Figures PATENTEU 3,905,869

SHEET 2 RE 3 20 ab in 5'0 60 TEMPERATURE ("0) R an I so

110 M m REF-M TEMPERATURE ("6) BACILLOPEPTIDASE C, A NEW ALKALINE PROTEASE AND ITS PRODUCTION BY CULTIVATING BACILLUS BACTERIA BACKGROUND SUMMARY OF THE INVENTION.

1. Fieldof The Invention This invention relates" to a novel and strong antiinflammatory alkaline protease named Bacillopeptidase C, and further relates to the fermentative production of this anti-inflammatory enzyme.

2. Prior Art Proteases have been used as digestives from the old day, and widely employed asanti-inflammatory agents, pus juice decomposing agents, detergents and food processing agents recently. Extensive investigations on this kind of enzyme have been made as their usage was developed and their demand increased. And, as a result, a number of novel and valuable proteases, in particular alkaline proteases, have been discovered.

The present inventors, now'satisfied with the functions and the productivity of the known alkaline proteases, have 'made extensive searches for proteaseproducing strains in the natural world and have confirmed that a strain of Bacillaceae produces a protease quite different in characteristics from the known alkaline proteases. The present invention was accomplished on the basis of this observation.

. DESCRlPTlON OF THE INVENTION The alkaline protease obtained in the present invention has physical and chemical'properties intrinsically different from those of the known alkaline proteases and displays exceedingly high anti-inflammatory and proteolytic activities. Having pronounced difference from the known alkaline proteases, the protease obtained according to the present invention has been characterized as a novel protease and thus nominated as Bacillopeptidase C. i

The conventional alkaline proteases are completely inactivated by about M of diisopropylfluorophos-- phatc (hereinafter refe'rred'to as DFP) which is a specific inhibitor against serine enzymes, while scarcely influenced by EDTA which is a specific inhibitor against neutral proteases. (L. Keay & B.S. Wildi, Biotech. and Bioeng. 12, 179 (1970); K. Morihara, Biochem. and Biophys. Research Communications 26, 656-657 1967 D. Tsuru et al., Arg; Biol. Chem, 30, 1266 (1966); K. Horikoshi Agr. Biol. chem. 35, 1407 1971 5 On the other hand, the novel protease of the present invention, Bacillopeptidase C, isconsidered as an entirely novel enzyme in that it is completely inactivated by both DFP and EDTA. It is very characteristic also in that it shows a marked inhibition rate in an antiinflammatory test according to the rat-limb edema method when carrageenin is injected as a phlogistic agent.

The strain which produces the novel protease Bacillopeptidase C according to the present invention is the one newly isolated from the soil by the present inven tors and is deposited in the Fermentation Research Institute of the Agency of Industrial Science and 'Technology, Chiba, Japan, under PERM-P No. 1522, and in the American Type Culture Collection, Rockville, Md, U.S.A. under ATCC. No. 21964. This strain is characterized as follows:

Vegetative rods, 0.7 to 0.9 by 2.5 to 3.5 microns. Motile by means of peritrichous flagella; Gram variable; spores, 1.3 to 1.6 microns spherical, terminal; optimum growth at pH 7 to 9; good growth at 28 to 40C; no growth at 50C; aerobic;

Colonies on meat extract agar: Smooth surface with lustre, opaque, and light yellowish brown in color.

Nutrientagar slants; Growth abundant, smooth.

Glucose nutrient agar slants: Growth equivalent to or better than nutrient agar slants;

I Soybean extract agar slantszSame as glucose nutrient agar slants, creamy;

Meat extractbroth containing NaCl: Growth in 5 percent NaCl, no growth in 10 percent NaCl;

Meat extract broth: Uniformly turbid, no bacterial film formed;

Meat extract gelatine stab: Strati form liquefaction at 22C within one week;

Litmus milk: Faintly pink in color;

Glucose asparagine agar: No growth;

Hydrolysis of starch: Negative;

Production of indole: Negative;

Production of acetylmethyl carbinol: Negative;

Catal'ase: Positive;

Liquefaction of gelatine: Positive;

Hydrolysis of casein: Positive;

Reduction of nitrate: Negative;

pH of glucose broth: pH 7.8 8.0;

Urea se Negative;

Reduction of Methylene Blue (dye): Positive;

Gas generation from nitrate under anaerobic condition: Negative;

Growth factor: Thiamine required;

Growth in glucose'broth under anaerobic condition: Negative.

A comparison of the microorganism of the present invention having the above described mycological properties with the known microorganisms disclosed in Bergeys Manual of Determinative Bacteriology, 7th Edition 1957, revealed that this strain is close to Bacillus brevis and to Bacillus sphaericus. But it differs from Bacillus sphaericus in the utilization of casein and gelatin and from Bacillus brevis in every respect of the morphology of spores, the growth in the medium incorporated with 5% NaCl and the requirement of Thiamine for growth. Thus no microorganism identical in all respects with the Bacillopeptidase C producing one of the present invention can be found among the known ones.

Accordingly, the strain used in the present invention has been named as a new strain, Bacillus sp. No. 794, belonging to Bacillaceae.

The novel protease, Bacillopeptidase C, is produced by cultivating in a medium a microorganism which be longs to Bacillaceae and is represented by Bacillus sp. No. 794.

The cultivation is performed, as a rule, in the usual manner adopted in the cultivation of general microorganisms, by either solid cultivation or liquid cultivation. Generally, liquid cultivation is preferable. Utilization cultivation medium are synthetic, semi-synthetic and natural media. As carbon source there are used glucose, sucrose, maltose, gluconic acid, starch, hydroly sates of starch and other carbohydrates, and as nitrogen source peptone, meat extract, corn steep liquor, Soybean meal, dry yeast, gluten, casein degradation product's, urea, ammonium sulfate and ammonium nitrate, singly or two or more in combination. Optionally, carbonate, sulfate or hydrochloride of magnesium, manganese or calcium and further sodium chloride or defoaming agent may be added in a suitable amount. The preferred cultivation temperature is 25-40C. The cultivation time depends upon the cultivation temperature, cultivation volume and cultivation system, but the production of the novel protease, Baciilopeptidasc C, is generally completed within 40 100 hours. Isolation of the produced protease from the medium can be carried out according to any of the conventional methods for the purification of enzymes. Namely, .those known methods such as filtration, salt-out by a water-soluble inorganic salt such as ammonium sulfate, sodium sulfate or magnesium sulfate, precipitation by the addition of a water miscible organic solvent such as methanol, ethanol, isopropanol or acetone, and adsorbance and elution using calcium phosphate, alumina, bentonite, exchange resin or ion-exhanger consisting of synthetic organic compound derived from polysaccharide dextran (commonly available under a trade name DEAE Sephadex A 50, a product ,of Pharmacia Co. Sweden.) can be employed in the present invention. The crude enzyme thus obtained is purified in the manner mentioned hereinafter to obtain an electrophorctically single and pure crystal.

The enzymological and physico-chemical properties of the novel protease, Bacillopeptidase C, of the present invention are shown as follows:

1. Function As shown in FIG. 1 of the drawings. the present enzyme is active at pH 6 l l on a milk casein substrate. The optimum pH is 9 93, indicating that this enzyme is an alkaline protease.

2. Substrate specificity The following Table 1 shows the substrate specificity of the Bacillopeptidase C.

Table l Substrates Relative initial velocity Milk casein I Albumin 40 Hemoglobin l l l Fihrinogcn 50 3. Measurement of enzyme activity The protease activity was measured according to the method based on the Anson & Hagiwaras (B.

Hagiwara et al., The J. of Biochem., 45, 185, 1958,

ibid. 45, 251, I958), which was slightly modified as fol lows: One ml. of the enzyme solution suitably diluted with M/50 CAPS buffer solution was mixed with ml. of 0.6% casein solution (pH 9.0) at 40C. After minutes incubation, the reaction was stopped by the addition of 5m]. of 0.44 M Trichloroacetic acid solution,

followed by minutes still incubation at C. Then,

2 ml. of 0.55 M sodium carbonate solution and 1 ml. 5 of Folin reagent were added and the mixture was kept still at 40C for 30 minutes. The optical density of the resulting mixture was measured at 660 my. against substrate blank. One enzyme activity unit was defined as the enzyme quantity capable of liberating peptide to such an amount that is equivalent in the said optical density to l ,u.g of tyrosin in a reaction mixture at pH 9.0, 40C in 1 minute.

4. Effects of pH on the activity and the stability of the enzyme.

The influence of pH value upon the activity of the present enzyme on a milk casein substrate is shown in FIG. I. The optimum pH is 9.3, and the activity is reduced to half at pH 6.5 7, or 10.5 11. FIG. 2 shows the influence of various pH values upon the stability of the enzyme, wherein the residual activity after treatment with M/ CAPS buffer solution (CAPS: Dotite reagent, cyclohexyl-aminopropanesulfonic acid) at a variety of pH values at 40C for 60 minutes was determined. As is apparent from FIG. 2 of the drawings, the enzyme is stable over a wide range of pH values; the residual activity of more than 90% exists in the range of pH 7 10.5.

5. Effect of temperature on the activity and the stability of the enzyme.

FIG. 3 of the drawings shows the effect of temperature on the enzyme activity. As seen in this figure, the optimum temperature for the activity of the present enzyme is around 45C, and more than 80% of activity is detectable in the range from 35C to C. As shown in FIG. 4 of the drawings, the enzyme is quite stable at below 40C and displays 50% remaining activity at C.

6. Conditions for inactivation due to pH and temper- 40 ature The enzyme is substantially inactivated at a pH value of below 6 and above l2, and completely inactivated through l-hour treatment at 40C at pH 4 or 12. It is also considerably unstable at pH 7 and 60C, and com- 4s pletcly inactivated at pH 7 and C, and likewise at pH 9.5 and C.

7. Influence of inhibitors Table 2 shows the influence of a variety of inhibiting substances. The numerical data are shown in terms of so the residual activity. Known alkaline proteases are serine enzymes containing serine in the active site and are generally known to be inhibited by specific inhibitors such as DFP and potato inhibitor but not inhibited by EDTA or o-phenanthroline. On the other hand, neutral 55 proteases are known to be metal enzymes and their behavior against the DEP and EDTA inhibitors is quite contrary to the serine enzymes.

Table 2 Inhibitors (on- Enzyme of T Enzyme A Enzyme ccntrations the Invention DFP l.l X l0(Mol) O(7() I00 ('7!) 7('71) DFP 1.] X I0 35 I00 2l Pl 0.l (mg/ml) 41 76 Pl 0.5 0 I00 18 TI ().l I00 I00 100 TI 0.0] I00 I00 I00 ED'TA l X 10 (M01) 0 0 I33 Table 2'-Continued Inhibitors Con- Enzyme of T Enzyme A Enzyme centrations the Invention EDTA l X 0 57 116 o-Phenanthroline l X 10' 95 0 93 PCMB 'i X 10" 100 110 PCMB 'i X 10"" I03 119 Legend):

DFP Diist)propylfluorophosphatc Pl Potato inhibitor Tl Trypsin inhibitor EDTA Ethylencdiamine letracctate PCMB i p-Chloromereuribenzoic acid The enzyme of the present invention is clearly a novel one; it is in one way a serine enzyme having serine at its active site as alkaline proteases do but in the other way obviously influenced by EDTA, indicating the important roles of bivalent metals in the enzyme action. For comparison, T enzyme and A enzyme were used as representatives of neutral protease and alkaline protease, respectively.

T enzyme: Thermolysin (Daiwa Kasei Kabushiki Kaisha), neutral protease, crystal A enzyme: Alkaline protease ll (Seikagaku Kogyo),

obtained from Bacillus subtilis var. amylosucc/zarilicus, crystallized three times 8. Method of purification The crude enzyme powder was dissolved in water to make a 10% solution thereof and the solution was stirred at 5C for several hours to completely extract the enzyme. Calcium acetate was added until the final concentration became about 1%. The pH value was adjusted to 7.5- 8.0 and the precipitated fraction was removed by filtration to obtain a supernatant liquid. Ammonium sulfate was further added until the final concentration reached 0.6 saturation. After allowing the supernatant liquid to stand for several hours, it was subjected to centrifugal separation to recover the precipitated fraction. Then, for the purpose of de-salting, it was again dissolved in M/200 of Tris hydrochloric acid buffer solution of pH 7.5 containing M/500 of calcium acetate, and subjected to dialysis against said buffer solution. Afterwards, it was subjected to a column of DEAE Sephadex A50 equilibrated by said buffer solution, and eluted with said buffer solution in an amount of 5 to 7 times as much as the bed volume, and then with the similar buffer solution containing 0.1 M NaCl to obtain a fraction containing the present enzymefRechromatography under the same condition raised the maximum specific activity of the enzyme up to top, followed by concentration to 1/10 to 1/15 volume to obtain the enzyme in crystal form.

9. Physical and chemical properties The novel protease, Bacillopeptidase C, is useful not only as an anti'inflammatory agent but also as a digestive, softener for leathers and edible meats, detergent, etc., by virtue of its high proteolitic activity.

The following is an explanation on the result of a test made to evaluate the antiinflammatory action of the novel protease, Bacillopeptidase C.

Each of four Donryu rats (weighed 130-150 g) was given intraperitoneally 0.5 mg per rat of each enzyme sample solution, and after 1 hour, 0.05 ml of an 1% carrageenin solution was given subcutaneously to the sole of one hind limb of each rat. After 3 hours, the extent of edema formed was measured by a slide caliper to assess the effect of each enzyme. The results showed that the edema inhibiting rate of trypsin was 33%, that of a-ehymotrypsin 25%, that of Bromeline 37%, while that of the enzyme of the invention was as high as (The numerical data indicate edema inhibiting rates according to rat-limb edema method wherein carrageenin is used the as phlogistic agent.)

From the above it is apparent that the novel protease, Bacillopeptidase C, has the above-mentioned properties that distinctly differ from the known proteases. Unlike the known proteases which are completely inactivated by DFP but not by ElDTA, the enzyme of the present invention is completely affected by both DP? and EDTA. In contrast to the known alkaline proteases having an isoelectric point of pH 8 or higher, the enzyme of the present invention has an isoelectric point of pH 6.0 and exibits far better anti-inflammatory activity. The Bacillopeptidase C is low in toxicity. Acute toxicity of the Bacillopeptidase C was tested by oral administration. LD50 0f the enzyme was calculated as 9500mg/kg (with confidence limits of 8796 10,260 mg/kg) in male mice and 8900 mg/kg (with 95% confidence limits of 8240.7- 9612 mg/kg) in female mice by the method of Litchfied & Wilcoxon (J. Pharmacol. Exp. Therap. 96, 99-1 13, 1949). The BacillopeptidaseC of this invention is, therefore, used as an effective anti-inflammatory agent. For use in the therapeutic treatment of inflammation and edema after surgery or trauma, 2.5 10 mg of the Bacillopeptidase C may be formulated into tablets or capsule with starch and/or lactose, and orally administered 2 to 3 of it for 3 to 4 times a day.

The following examples illustrate the invention with out limiting its scope.

EXAMPLE 1 Twenty liters of a medium containing 1% of starch, 2% of soybean oil cake, 1% of wheat bran, 0.5% of potassium phosphate, 0.5% of calcium carbonate, and 0.05% of magnesium sulfate was charged into a jar fermenter of 30 liter volume and sterilized at 120C for 20 minutes under pressure. The medium was then inoculated with 200 ml of a seed culture of the Bacillus No. 794 (identified as ATCC No. 21964 or PERM-P No. 1522), which had been cultivated separately in a neutrient broth. Cultivation was performed with stirring at 300 rpm at 30C for 3 days under 100% aeration. Immediately after the cultivation was completed, the cultures were subjected to a centrifugal separation to remove the bacterium and there was obtained liters of the filtrate. The filtrate was then concentrated to 3 liters under reduced pressure and then its pH value was adjusted to 8.0 by addition of 27 g of calcium acetate. The precipitate formed was removed by filtration and the filtrate was saturated with ammonium sulfate up to 0.6 saturation and subjected to centrifugal separation to recover the precipitate formed. 80% of the activity at the completion of the fermentation was thus recovered. The precipitate was again dissolved in 300 ml of a solution containing M/500 of calcium acetate in M/200 of Tris hydrochloric acid buffer solution of pH 7.5 and the solution was then dialyzed against said buffer solution. The resulting enzyme solution was adsorbed on a column of 3 liters of DEAE Sephadex A50 equilibrated with said buffer solution and washed with about 10 liters of similar buffer solution, and then with the same buffer solution containing 0.1 NaCl whereupon the enzyme of the invention was eluted. In 3 liters of the eluate, about 70% activity was recovered. Re-chromatography of the eluate under the same condition and concentration to its 1/10 volume gave 0.8 g of crystal of the Bacillopeptidase C.

EXAMPLE 2 Two liters of the culture filtrate obtained as described in Example 1 was subjected without being concentrated to a salting-out operation with ammonium sulfate, then to dialysis and finally to chromatography using DEAE-Sephadex. The eluate obtained by a concentration gradient elution method was concentrated to its 1 /8 volume to obtain 60 mg of crystal of the Bacillopeptidase C.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a graph showing the active pH range of Bacillopeptidase C, FIG. 2 a graph showing the stable pH range thereof, FIG. 3 a graph showing the active temperature, range thereof, FIG. 4 a graph showing the stable temperature range and FIG. 5 a graph showing the isolation condition when using a 8 ml column of DEAS Sephadex A-50.

What we claim is:

1. Process for the production of the novel alkaline protease enzyme named Bacillopeptidase C which is an effective anti-inflammatory agent, having the following properties:

1. elementary analysis C 51.22%,

0.99%, N 16.99%, ash 1.62%, 2. molecular weight about 27,000,

3. maximum absorption in its ultraviolet absorption spectrum at a wavelength of 280 millimicrons E 12.7 (pH 6.8),

4. isoelectric point: pH 6.0,

5. specific activity 1 1,400 ,u/mg N,

6. optimal activity at a pH from 9.0 to 9.3 as indicated in FIG. 1, at a temperature about 45C as indicated in FIG. 3,

7. stability in the pH range of 7 to 10.5 as indicated in FIG. 2,

8. inhibition by ethylene-diamine tetraacetate, diisopropyl fluorophosphate or potato inhibitor but not inhibited by trypsin inhibitor, o-phenanthroline or p-chloromercuribenzoicl acid,

which process comprises a. cultivating Bacillus sp. No. 794 (ATCC 21964 or PERM-P No. 1522) in a production medium until the said enzyzme is substantially accumulated in the medium, and

b. recovering the resulting Bacillopeptidase C from the medium.

2. Process according to claim 1, wherein the production medium is solid.

3. Process according to claim 1, wherein the production medium is liquid.

4. Process according to claim 1, wherein the production medium is selected from the group consisting of natural, semi-synthetic and synthetic media.

5. Process according to claim 1, wherein the production medium contains at least one member selected from the group consisting of glucose, sucrose, maltose, gluconic acid, starch, hydrolysates of starch and other carbohydrates.

6. Process according to claim 1, wherein the production medium contains at least one member selected from the group consisting of peptonc, meat extract, corn steep liquor, soybean meal, dry yeast, gluten, casein degradation products, urea, ammonium sulfate and ammonium nitrate.

7. Process according to claim 1, wherein the production medium contains a. a salt of magnesium, manganese or calcium consisting of a carbonate, sulfate or hydrochloride;

b. sodium chloride; and

c. defoaming agent.

8. Process according to claim 1, wherein the cultivating takes place at a temperature from about 25 to about 40C.

9. Process according to claim 1, wherein the cultivating is carried out within from about 40 to about hours.

10. A novel alkaline protease named Bacillopeptidase C, which is an effective anti-inflammatory agent, and which is produced by a process of (a) cultivating Bacillus sp. No. 794 (ATCC 21964 or FERM-P No. 1522) in a production medium until the said enzyme is substantially accumulated in the medium, and (b) recovering the resulting Bacillopeptidase C from the medium and which has the following properties:

1. elementary analysis C 51.22%, H 6.92%, S

0.99%, N 16.99%, ash 1.62%;

2. molecular weight about 27,000;

3. maximum absorption in its ultraviolet absorption spectrum at a wavelength of 280 millimicrons E,,.,,,""" 12.7 (pH 6.8);

4. isoelectric point pH 6.0;

5. specific activity 1 1,400 umg N;

9 l 6. optimal activity at a pH from 9.0 to 9.3 as indi- 8. inhibition by ethylene-diamine tetraacetate, diisocated in FIG. 1, at a temperature about 45C as inpropyl fluorophosphate or potato inhibitor but not dicated in FIG. 3; inhibited by trypsin inhibitor, o-phenanthroline or 1. stability in the pH range of 7 to 10.5 as indicated p-chloromercuribenzoic acid].

inFIG.2; 

1. elementary analysis : C 51.22%, H 6.92%, S 0.99%, N 16.99%, ash 1.62%;
 1. stability in the pH range of 7 to 10.5 as indicated in FIG. 2;
 1. elementary analysis : C 51.22%, H 6.92%, S 0.99%, N 16.99%, ash 1.62%,
 1. Process for the production of the novel alkaline protease enzyme named Bacillopeptidase C which is an effective anti-inflammatory agent, having the following properties:
 1. STABILITY IN THE PH RANGE OF 7 TO 10.5 AS INDICATED IN FIG. 2,
 1. ELEMNTARY ANALYSIS : C51.22%, H 6.92%, S 0.99%, N 16.99%, ASH 1.62%,
 2. MOLECULAR WEIGHT : ABOUT 27,000,
 2. molecular weight : about 27,000,
 2. molecular weight : about 27,000;
 2. Process according to claim 1, wherein the production medium is solid.
 3. Process according to claim 1, wherein the production medium is liquid.
 3. maximum absorption in its ultraviolet absorption spectrum at a wavelength of 280 millimicrons E1cm1% : 12.7 (pH 6.8);
 3. MAXIMUM ABSORPTION IN ITS ULTRAVIOLET ABSORPTION SPECTRUM AT A WAVELENGTH OF 280 MILLIMICRONS E1CM% : 12.7 (PH 6.8),
 3. maximum absorption in its ultraviolet absorption spectrum at a wavelength of 280 millimicrons E1cm 1% : 12.7 (pH 6.8),
 4. isoelectric point: pH 6.0,
 4. ISOELECTRIC POINT : PH 6.0,
 4. isoelectric point : pH 6.0;
 4. Process according to claim 1, wherein the production medium is selected from the group consisting of natural, semi-synthetic and synthetic media.
 5. Process according to claim 1, wherein the production medium contains at least one member selected from the group consisting of glucose, sucrose, maltose, gluconic acid, starch, hydrolysates of starch and other carbohydrates.
 5. specific activity : 11,400 Mu mg N;
 5. SPECIFIC ACTIVITY : 11,400 UMG N,
 5. specific activity : 11,400 Mu /mg N,
 6. optimal activity at a pH from 9.0 to 9.3 as indicated in FIG. 1, at a temperature about 45*C as indicated in FIG. 3,
 6. OPTIMAL ACTIVITY AT A PH FROM 9.0 TO 9.3 AS INDICATED IN FIG. 1, AT A TEMPERATURE ABOUT 45*C AS INDICATED IN FIG. 3,
 6. optimal activity at a pH from 9.0 to 9.3 as indicated in FIG. 1, at a temperature about 45*C as indicated in FIG. 3;
 6. Process according to claim 1, wherein the production medium contains at least one member selected from the group consisting of peptone, meat extract, corn steep liquor, soybean meal, dry yeast, gluten, casein degradation products, urea, ammonium sulfate and ammonium nitrate.
 7. Process according to claim 1, wherein the production medium contains a. a salt of magnesium, manganese or calcium consisting of a carbonate, sulfate or hydrochloride; b. sodium chloride; and c. defoaming agent.
 7. stability in the pH range of 7 to 10.5 as indicated in FIG. 2,
 8. inhibition by ethylene-diamiNe tetraacetate, diisopropyl fluorophosphate or potato inhibitor but not inhibited by trypsin inhibitor, o-phenanthroline or p-chloromercuribenzoicl acid, which process comprises a. cultivating Bacillus sp. No. 794 (ATCC 21964 or FERM-P No. 1522) in a production medium until the said enzyzme is substantially accumulated in the medium, and b. recovering the resulting Bacillopeptidase C from the medium.
 8. inhibition by ethylene-diamine tetraacetate, diisopropyl fluorophosphate or potato inhibitor but not inhibited by trypsin inhibitor, o-phenanthroline or p-chloromercuribenzoic acid.
 8. Process according to claim 1, wherein the cultivating takes place at a temperature from about 25* to about 40*C.
 8. INHIBITION BY ETHYLENE-DIAMINE TETRAACETATE, DIISOPROPYL FLUOROPHOSPHATE OR POTATO INHIBITOR BUT NOT INHIBITED BY TRYPSIN INHIBITOR, O-PHENANTHROLINE OR P-CHLOROMERCURIBENZOIC ACID.
 9. Process according to claim 1, wherein the cultivating is carried out within from about 40 to about 100 hours.
 10. A NOVEL ALKLINE PROTEASE NAMED BACILLOPEPTIDASE C, WHICH IS AN EFFECTIVE ANTI-INFLAMMATORY AGENT, AND WHICH IS PRODUCED BY A PROCESS OF (A) CULTIVATING BACILLUS SP. NO. 794 (ATCC 21964 OR FERM-P NO. 1522) IN A PRODUCTION MEDIUM UNTIL THE SAID ENZYME IS SUBSTANTIALLY ACCUMULATED IN THE MEDIUM, AN (B) RECOVERING THE RESULTING BACILLOPEPTIDASE C FROM THE MEDIUM AND WHICH HAS THE FOLLOWING PROPERTIES: 